Avery Solco

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells, indicative of alternative macrophage activation

ETHNOPHARMACOLOGICAL RELEVANCE The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages. AIM OF THE STUDY In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity. MATERIALS AND METHODS Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/- LPS. RESULTS Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression. CONCLUSIONS These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concomitantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling.

Endogenous Levels of Echinacea Alkylamides and Ketones Are Important Contributors to the Inhibition of Prostaglandin E2 and Nitric Oxide Production in Cultured Macrophages

Because of the popularity of Echinacea as a dietary supplement, researchers have been actively investigating which Echinacea constituent or groups of constituents are necessary for immune-modulating bioactivities. Our prior studies indicate that alkylamides may play an important role in the inhibition of prostaglandin E2 (PGE(2)) production. High-performance liquid chromatography fractionation, employed to elucidate interacting anti-inflammatory constituents from ethanol extracts of Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, and Echinacea tennesseensis, identified fractions containing alkylamides and ketones as key anti-inflammatory contributors using lipopolysaccharide-induced PGE(2) production in RAW264.7 mouse macrophage cells. Nitric oxide (NO) production and parallel cytotoxicity screens were also employed to substantiate an anti-inflammatory response. E. pallida showed significant inhibition of PGE(2) with a first round fraction, containing gas chromatography-mass spectrometry (GC-MS) peaks for Bauer ketones 20, 21, 22, 23, and 24, with 23 and 24 identified as significant contributors to this PGE(2) inhibition. Chemically synthesized Bauer ketones 21 and 23 at 1 microM each significantly inhibited both PGE(2) and NO production. Three rounds of fractionation were produced from an E. angustifolia extract. GC-MS analysis identified the presence of Bauer ketone 23 in third round fraction 3D32 and Bauer alkylamide 11 making up 96% of third round fraction 3E40. Synthetic Bauer ketone 23 inhibited PGE(2) production to 83% of control, and synthetic Bauer alkylamide 11 significantly inhibited PGE(2) and NO production at the endogenous concentrations determined to be present in their respective fraction; thus, each constituent partially explained the in vitro anti-inflammatory activity of their respective fraction. From this study, two key contributors to the anti-inflammatory properties of E. angustifolia were identified as Bauer alkylamide 11 and Bauer ketone 23.

Alkamide production from hairy root cultures of Echinacea

Hairy root cultures of Echinacea, one of the most important medicinal plants in the US, represent a valuable alternative to field cultivation for the production of bioactive secondary metabolites. In this study, the three most economically important species of Echinacea (Echinacea purpurea, Echinacea pallida, and Echinacea angustifolia) were readily transformed with two strains of Agrobacterium that produce the hairy root phenotype. Transformed roots of all three species exhibited consistent accelerated growth and increased levels of alkamide production. Optimization of the culture of Echinacea hairy roots was implemented to enhance both growth and alkamide production concomitantly. The use of half-strength Gamborg’s B5 medium supplemented with 3.0% sucrose was twice as effective in maintaining hairy root production than any other media tested. The addition of indolebutyric acid increased the growth rate of roots by as much as 14-fold. Alkamide production increased severalfold in response to the addition of the elicitor, jasmonic acid, but did not respond to the addition of indolebutyric acid. Induced accumulation of the important bioactive compounds, alkamides 2 and 8, was observed both in transformed roots and in response to jasmonic acid treatments. The results of this study demonstrate the efficacy of hairy root cultures of Echinacea for the in vitro production of alkamides and establish guidelines for optimum yield.